Systems and Methods for Analyzing Nucleic Acid Sequences

Abstract

The invention relates to systems and methods for analyzing clinically relevant nucleic acid sequences.

1 . A system for performing an assay on a biological sample, comprising:
 (a) a central controller programmed to:
 (i) exchange information about the biological sample with an outside system or database; and 
 (ii) exchange information about the biological sample with one or more modules of the system; 
   (b) a sample transfer module for transferring a portion of the sample to a first container;   (c) a nucleic acid extraction module for extracting nucleic acids from cells within the portion and for transferring the portion from the first container to a second container;   (d) a nucleic acid measurement module for measuring the concentration of nucleic acids in the portion;   (e) a PCR preparation module for adding polymerase chain reaction (PCR) reaction materials to the portion;   (f) a thermocyling module for amplifying a target sequence and extending a primer in the portion;   (g) a primer extension preparation module for adding primer extension reaction materials to the portion;   (h) a mass spectrometry preparation module for removing a sample of the portion from the second container to a support for analysis by mass spectrometry; and   (i) a mass spectrometry module for analyzing the sample.   
     
     
         2 . The system of  claim 1 , further comprising linking software that enables the central controller to communicate with at least one other module in the system. 
     
     
         3 . The system of  claim 1 , further comprising a plate editor module that provides sample information to the PCR preparation module. 
     
     
         4 . The system of  claim 1 , further comprising: (j) a transport module comprising one or more robotic arms or tracks to transport a biological sample, or portion thereof, between at least two module of (a) to (i), and arranged to receive information from and transmit information to the central controller. 
     
     
         5 . The system of  claim 1 , further comprising: (j) a detection module for detecting the presence of a sample and monitoring the progress of the sample through the system, and arranged to receive information from and transmit information to the central controller. 
     
     
         6 . The system of  claim 1 , wherein the PCR preparation system includes a primer set selected from the group consisting of: SEQ ID NOS:1, 2, and 3; SEQ ID NOS:4, 5 and 6; SEQ ID NOS:7, 8, and 9; and SEQ ID NOS:10, 11, and 12; SEQ ID NOS:13, 14, and 15; SEQ ID NOS: 16, 17 and 18; SEQ ID NOS:19, 20, and 21; and SEQ ID NOS:22, 23 and 24; SEQ ID NOS:25, 26, and 27; SEQ ID NOS:28, 29, and 30; SEQ ID NOS:31, 32, and 33; SEQ ID NOS:34, 35 and 36; SEQ ID NOS:37, 38 and 39; SEQ ID NOS:40, 41 and 42; SEQ ID NOS:43, 44 and 45; SEQ ID NOS:46, 47 and 48; SEQ ID NOS:49, 50 and 51; SEQ ID NOS:52, 53 and 54; SEQ ID NOS: 55, 56 and 57; SEQ ID NOS:58, 59 and 60; SEQ ID NOS: 61, 62, and 63; SEQ ID NOS: 64, 65, and 66; SEQ ID NOS: 67, 68, and 69; SEQ ID NOS: 70, 71, and 72; SEQ ID NOS: 73, 74, and 75; SEQ ID NOS: 76, 77, and 78; SEQ ID NOS: 79, 80, and 81; SEQ ID NOS: 82, 83, and 84; SEQ ID NOS: 85, 86, and 87; SEQ ID NOS: 88, 89, and 90; SEQ ID NOS: 91, 92, and 93; SEQ ID NOS: 94, 95, and 96; SEQ ID NOS: 97, 98, and 99; SEQ ID NOS: 100, 101, and 102; SEQ ID NOS: 103, 104, and 105; SEQ ID NOS: 106, 107, and 108; SEQ ID NOS: 109, 110, and 111; SEQ ID NOS: 112, 113, and 114; SEQ ID NOS: 115, 116, and 117; SEQ ID NOS: 118, 119, and 120; SEQ ID NOS: 121, 122, and 123; SEQ ID NOS: 124, 125, and 126; SEQ ID NOS: 127, 128, and 129; SEQ ID NOS: 130, 131, and 132; SEQ ID NOS: 133, 134, and 135; SEQ ID NOS: 136, 137, and 138; SEQ ID NOS: 139, 140, and 141; SEQ ID NOS: 142, 143, and 144; SEQ ID NOS: 145, 146, and 147; SEQ ID NOS: 148, 149, and 150; SEQ ID NOS: 151, 152, and 153; SEQ ID NOS: 154, 155, and 156; SEQ ID NOS: 157, 158, and 159; SEQ ID NOS: 160, 161, and 162; SEQ ID NOS: 163, 164, and 165; SEQ ID NOS: 166, 167, and 168; SEQ ID NOS: 169, 170, and 171; SEQ ID NOS: 172, 173, and 174; SEQ ID NOS: 175, 176, and 177; SEQ ID NOS: 178, 179, and 180; SEQ ID NOS: 181, 182, and 183; SEQ ID NOS: 184, 185, and 186; SEQ ID NOS: 187, 188, and 189; SEQ ID NOS: 190, 191, and 192; SEQ ID NOS: 193, 194, and 195; SEQ ID NOS: 196, 197, and 198; SEQ ID NOS: 199, 200, and 201; SEQ ID NOS: 202, 203, and 204; SEQ ID NOS: 205, 206, and 207; SEQ ID NOS: 208, 209, and 210; SEQ ID NOS: 211, 212, and 213; SEQ ID NOS: 214, 215, and 216; SEQ ID NOS: 217, 218, and 219; SEQ ID NOS: 220, 221, and 222; SEQ ID NOS: 223, 224, and 225; SEQ ID NOS: 226, 227, and 228; SEQ ID NOS: 229, 230, and 231; SEQ ID NOS: 232, 233, and 234; SEQ ID NOS: 235, 236, and 237; SEQ ID NOS: 238, 239, and 240; SEQ ID NOS: 241, 242, and 243; SEQ ID NOS: 244, 245, and 246; SEQ ID NOS: 247, 248, and 249; SEQ ID NOS: 250, 251, and 252; SEQ ID NOS: 253, 254, and 255; SEQ ID NOS: 256, 257, and 258; SEQ ID NOS: 259, 260, and 261; SEQ ID NOS: 262, 263, and 264; SEQ ID NOS: 265, 266, and 267; SEQ ID NOS: 268, 269, and 270; SEQ ID NOS: 271, 272, and 273; SEQ ID NOS: 274, 275, and 276; SEQ ID NOS: 277, 278, and 279; SEQ ID NOS: 280, 281, and 282; SEQ ID NOS: 283, 284, and 285; SEQ ID NOS: 286, 287, and 288; SEQ ID NOS: 289, 290, and 291; SEQ ID NOS: 292, 293, and 294; SEQ ID NOS: 295, 296, and 297; SEQ ID NOS: 298, 299, and 300; SEQ ID NOS: 301, 302, and 303; SEQ ID NOS: 304, 305, and 306; SEQ ID NOS: 307, 308, and 309; SEQ ID NOS: 310, 311, and 312; SEQ ID NOS: 313, 314, and 315; SEQ ID NOS: 316, 317, and 318; SEQ ID NOS: 319, 320, and 321; SEQ ID NOS: 322, 323, and 324; SEQ ID NOS: 325, 326, and 327; SEQ ID NOS: 328, 329, and 330; SEQ ID NOS: 331, 332, and 333; SEQ ID NOS: 334, 335, and 336; SEQ ID NOS: 337, 338, and 339; SEQ ID NOS: 340, 341, and 342; SEQ ID NOS: 343, 344, and 345; SEQ ID NOS: 346, 347, and 348; SEQ ID NOS: 349, 350, and 351; SEQ ID NOS: 352, 353, and 354; SEQ ID NOS: 355, 356, and 357; SEQ ID NOS: 358, 359, and 360; SEQ ID NOS: 361, 362, and 363; SEQ ID NOS: 364, 365, and 366; SEQ ID NOS: 367, 368, and 369; SEQ ID NOS: 370, 371, and 372; SEQ ID NOS: 373, 374, and 375; SEQ ID NOS: 376, 377, and 378; SEQ ID NOS: 379, 380, and 381; SEQ ID NOS: 382, 383, and 384; SEQ ID NOS: 385, 386, and 387; SEQ ID NOS: 388, 389, and 390; SEQ ID NOS: 391, 392, and 393; SEQ ID NOS: 394, 395, and 396; SEQ ID NOS: 397, 398, and 399; SEQ ID NOS: 400, 401, and 402; SEQ ID NOS: 403, 404, and 405; SEQ ID NOS: 406, 407, and 408; SEQ ID NOS: 409, 410, and 411; SEQ ID NOS: 412, 413, and 414; SEQ ID NOS: 415, 416, and 417; SEQ ID NOS: 418, 419, and 420; SEQ ID NOS: 421, 422, and 423; SEQ ID NOS: 424, 425, and 426; SEQ ID NOS: 427, 428, and 429; SEQ ID NOS: 430, 431, and 432; SEQ ID NOS: 433, 434, and 435; SEQ ID NOS: 436, 437, and 438; SEQ ID NOS: 439, 440, and 441; SEQ ID NOS: 442, 443, and 444; SEQ ID NOS: 445, 446, and 447; SEQ ID NOS: 448, 449, and 450; SEQ ID NOS: 451, 452, and 453; SEQ ID NOS: 454, 455, and 456; SEQ ID NOS: 457, 458, and 459; SEQ ID NOS: 460, 461, and 462; SEQ ID NOS: 463, 464, and 465; SEQ ID NOS: 466, 467, and 468; SEQ ID NOS: 469, 470, and 471; SEQ ID NOS: 472, 473, and 474; SEQ ID NOS: 475, 476, and 477; SEQ ID NOS: 478, 479, and 480; SEQ ID NOS: 481, 482, and 483; SEQ ID NOS: 484, 485, and 486; SEQ ID NOS: 487, 488, and 489; SEQ ID NOS: 490, 491, and 492; SEQ ID NOS: 493, 494, and 495; SEQ ID NOS: 496, 497, and 498; SEQ ID NOS: 499, 500, and 501; and SEQ ID NOS: 502, 503, and 504, each primer set including two amplification primers and one detection extension primer. 
     
     
         7 . The system of  claim 1 , wherein the central controller is a personal computer system. 
     
     
         8 . The system of  claim 1 , wherein the central controller includes linking software. 
     
     
         9 . The system of  claim 1 , wherein the sample transfer module includes a pipetting robot. 
     
     
         10 . The system of  claim 1 , wherein the nucleic acids measurement module includes an ultraviolet light spectrophotometer or a fluorometer. 
     
     
         11 . The system of  claim 1 , wherein the PCR preparation module includes a pipetting robot. 
     
     
         12 . The system of  claim 1 , wherein the thermocycling module includes a thermocyler. 
     
     
         13 . The system of  claim 1 , further comprising a computer-readable medium comprising one or more programs for instructing a given module. 
     
     
         14 . The system of  claim 1 , wherein the support is a chip or microwell. 
     
     
         15 - 16 . (canceled) 
     
     
         17 . A method of performing a diagnostic assay on a biological sample, the method comprising:
 (a) receiving a biological sample, generating information about the biological sample, and transmitting the information to a central controller;   (b) transferring a portion of the biological sample to a first container;   (c) extracting nucleic acids from cells within the portion and transferring the portion to a second container;   (d) measuring the concentration of extracted nucleic acids in the portion;   (e) adding polymerase chain reaction (PCR) materials to the portion;   (f) amplifying target nucleic acids in the portion;   (g) adding primer extension reaction materials to the portion;   (h) extending a detection extension primer in the portion;   (i) transferring a sample of the portion from the second container to a support;   (j) analyzing the sample and exporting data to the central controller using a mass spectrometry system; and   (k) transmitting the data from the central controller to an output device, external system, or database.   
     
     
         18 . The method of  claim 17 , wherein at steps (a) to (k) are performed automatically by an automated system. 
     
     
         19 . The method of  claim 18 , wherein the automated system includes at least one component selected from the group consisting of: a central controller, a sample transfer module, a nucleic acid extraction module, a nucleic acid measurement module, a PCR preparation module, a thermocyling module, a primer extension preparation module, a mass spectrometry preparation module, and a mass spectrometry module. 
     
     
         20 . The method of  claim 17 , wherein the diagnostic assay is an assay for detecting mutations in a gene. 
     
     
         21 . The method of  claim 20 , wherein the gene is selected from the group consisting of: 5,10-Methylenetetrahydrofolate Reductase (MTFR); Coagulation Factor II; Coagulation Factor V; hemochromatosis (HFE); and a glucocerebrosidase (GC). fibroblast growth factor receptor 3; aspartoacylase; Glucocerebrosidase; Coagulation Factor VII; Fanconi Anemia, Complementation Group C (FANCC); inhibitor of kappa light polypeptide gene enhancer in b cells, kinase complex-associated protein; acid sphingomyelinase; hexosaminidase; angiotensin i-converting enzyme; adenylate cyclase 9; apolipoprotein A-1; apolipoprotein E; endothelial leukocyte adhesion molecule 1; fc fragment of IGG, low affinity IIa, receptor; fibrinogen beta chain; coagulation factor II, factor XIII; guanine nucleotide-binding protein beta-3; integrin, alpha-2, glycoprotein Ia/Iia; glycoprotein Ib, platelet, alpha polypeptide; intercellular adhesion molecule 1; glycoprotein Ia/IIa (a2), integrin, alpha-2; platelet glycoprotein Iib, integrin, alpha-2b; glycoprotein IIb/IIIa, integrin, beta-3,3-hydroxy-3-methylglutaryl-coa reductase; lymphocyte adhesion molecule 1; methylene tetrahydrofolate reductase; plasminogen activator inhibitor 1; platelet alpha-granule membrane protein; transforming growth factor-beta receptor, type III; thrombomodulin; tumor necrosis factor; vascular cell adhesion molecule; coagulation factor II receptor; glycoprotein VI, platelet; purinergic receptor P2Y, g protein-coupled, 1; purinergic receptor P2Y, G protein-coupled, 12; prostaglandin-endoperoxide synthase 1; prostaglandin-endoperoxide synthase 2; thromboxane A2 receptor, platelet; and thrombospondin I. 
     
     
         22 . The method of  claim 17 , wherein the diagnostic assay is an assay for detecting a pathogen in the sample. 
     
     
         23 . The method of  claim 22 , wherein the pathogen is a virus, bacterium, or fungus. 
     
     
         24 . The method of  claim 23 , wherein the virus is a virus of the family Herpesviridae. 
     
     
         25 . The method of  claim 24 , wherein the virus is of the genus cytomegalovirus (CMV). 
     
     
         26 . An automated method for detecting mutations in a target gene, the method comprising:
 a) amplifying a target sequence using PCR and automatically performing a primer extension reaction using a set of three primers, each set of primers including two amplification primers and one detection extension primer;   b) automatically transferring detection extension primers to a mass spectrometry device; and   c) automatically determining the molecular weights of the detection extension primers by mass spectrometry following the primer extension reaction, wherein a change in the molecular weight of the extended primer, as compared to a control, indicates the presence of a mutation in the gene.   
     
     
         27 . The method of  claim 26 , further comprising automatically transmitting information related to the presence of the mutation to a central controller. 
     
     
         28 . The method of  claim 26 , wherein the gene is a 5,10-Methylenetetrahydrofolate Reductase (MTFR) gene, and the set of three primers is selected from the group consisting of: SEQ ID NOS: 1, 2, and 3; SEQ ID NOS: 4, 5 and 6; SEQ ID NOS: 7, 8, and 9; and SEQ ID NOS: 10, 11, and 12; each set of primers including two amplification primers and one detection extension primer. 
     
     
         29 . The method of  claim 26 , wherein the gene is a Coagulation Factor II gene, and the set of three primers is selected from the group consisting of: SEQ ID NOS: 13, 14, and 15 and SEQ ID NOS: 16, 17 and 18; each primer set including two amplification primers and one detection extension primer. 
     
     
         30 . The method of  claim 26 , wherein the gene is a Coagulation Factor Vgene, and the set of three primers is selected from the group consisting of: SEQ ID NOS: 19, 20, and 21 or SEQ ID NOS: 22, 23 and 24; each primer set including two amplification primers and one detection extension primer. 
     
     
         31 . The method of  claim 26 , wherein the gene is a hemochromatosis (HFE) gene, and the set of three primers is selected from the group consisting of: SEQ ID NOS: 40, 41, and 42, SEQ ID NOS: 43, 44 and 45; SEQ ID NOS: 46, 47 and 48; SEQ ID NOS: 49, 50 and 51; SEQ ID NOS: 52, 53 and 54; or SEQ ID NOS: 55, 56 and 57; each set of primers including two amplification primers and one detection extension primer. 
     
     
         32 . An automated method for detecting a pathogen in a biological sample, the method comprising:
 a) amplifying a target sequence using PCR and automatically performing a primer extension reaction using a set of three primers, each set of primers including two amplification primers and one detection extension primer;   b) automatically transferring detection extension primers to a mass spectrometry device; and   c) automatically determining the molecular weights of the detection extension primers by mass spectrometry following the primer extension reaction, wherein a change in the molecular weight of the extended primer, as compared to controls, indicates the presence of a pathogen in the sample.   
     
     
         33 . The method of  claim 32 , wherein the controls include an internal control for determining the amount of the pathogen in the sample. 
     
     
         34 . The method of  claim 32 , wherein the pathogen is cytomegalovirus (CMV), and the three primers are selected from the group consisting of: SEQ ID NOS: 25, 26, and 27; SEQ ID NOS: 28, 29 and 30; SEQ ID NOS: 31, 32, and 33; SEQ ID NOS: 34, 35, and 36; SEQ ID NOS: 37, 38, and 39; and SEQ ID NOS: 58, 59, and 60; each primer set including two amplification primers and one detection extension primer. 
     
     
         35 - 36 . (canceled) 
     
     
         37 . A computer readable medium comprising a program for instructing a central controller in an automated system for performing an assay on a biological sample to:
 (a) receive a biological sample, generate information about the biological sample, and transmit the information into a central controller;   (b) transfer a portion of the biological sample to a first container;   (c) extract nucleic acids from cells within the portion and transfer the portion to a second container;   (d) measure the concentration of extracted nucleic acids in the portion;   (e) add polymerase chain reaction (PCR) materials to the portion;   (f) amplify target nucleic acids in the portion;   (g) add primer extension reaction materials to the portion;   (h) extend a detection extension primer in the portion;   (i) transfer a sample of the portion from the second container to a support;   (j) analyze the sample and exporting data to the central controller using a mass spectrometry system; and   (k) transmit the data from the central controller to an output device, external system, or database.